RESUMO
Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and 'mock-transfected' leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.
Assuntos
Expressão Gênica/efeitos dos fármacos , Leishmania donovani , Metionina Adenosiltransferase , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , S-Adenosilmetionina/biossíntese , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Técnicas de Cultura de Células , Clonagem Molecular , Cicloeximida/farmacologia , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Cinética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Leupeptinas/farmacologia , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Oligopeptídeos/farmacologia , Plasmídeos , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/genética , TransfecçãoRESUMO
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.